1. Field of the Invention
The present invention relates to the field of fluorescence immunoassays useful in the determination of analytes in fluid samples.
2. Description of the Background Art
There are several known methods for detecting analytes (e.g., hormones, enzymes, other proteins, therapeutic agents, drugs of abuse, etc.) in liquid samples such as biological fluids. Among the known types of methods, immunoassays have emerged as sensitive techniques for determining minute amounts of certain organic compounds. Immunoassay methods generally are based on the ability of a receptor molecule, usually an antibody, to specifically recognize a particular spatial and/or polar organization of a ligand molecule, and thereby selectively bind to the ligand molecule.
Certain of the known immunoassay techniques involve the use of fluorophore molecules, which are able to absorb light a one wavelength and emit light at another wavelength. For example, U.S. Pat. No. 4,272,505 to Smith describes a method for assaying a biological fluid sample for a thyroid hormone. This method is based on the principle of fluorescence suppression of a fluorophore by thyroid hormone. The Smith method is a competitive-type assay involving formation of a mixture of a fluid sample with a known amount of fluorophore-labeled thyroid hormone, the fluorophore having a fluorescence level which is substantially suppressed by the thyroid hormone to which the fluorophore is bound. Antibody capable of binding to the fluorophore-labeled thyroid hormone as well as thyroid hormone present in the sample is introduced into the mixture. The antibody is thought to sterically alter the labeled thyroid hormone, thus changing the degree of suppression of the fluorescence of the fluorophore bound thereto. The fluorescence level of the mixture then is measured and the amount of thyroid hormone in the sample is calculated by comparing the fluorescence level of the mixture with a standard fluorescence level.
U.S. Pat. No. 4,133,873 to Noller discloses a method for determining the amount of a member of a group consisting of an extracellular antigen and an extracellular antibody capable of specifically combining with said antigen. The method involves tagging the member with a fluorophore and exposing the tagged member to a pulse of light of a first wavelength sufficient to cause emission by the tagged exposed member of secondary light having a second wavelength different from the first wavelength. The secondary light is sensed to generate a perceptible signal in response to and commensurate with the sensed secondary light.
An immunoassay utilizing two different ligands tagged with separate fluorophores which independently fluoresce at different wavelengths is disclosed in U.S. Pat. No. 4,385,126 to Chen et al. The two tagged ligands are capable of immunologically binding to each other, and the two different ligands may be detected independently through their independent tagging constituents (fluorophores) for quality control, internal calibration (standardization), determination of viability and shelf life, and the like.
U.S. Pat. Nos. 3,996,345, 4,174,384, 4,199,559 and 4,261,968, all to Ullman et al., disclose immunoassays employing antibodies and a fluorescer-quencher chromophoric pair. The methods are based on the phenomenon of energy transfer between two chromophores which form a fluorescer-quencher pair. The methods involve irradiation of a fluorescer molecule with light of a first wavelength which is absorbed by the fluorescer and resultant emission of light of a longer wavelength by the fluorescer. If a quencher chromophore is within less than about 100 .ANG. of the fluorescer and absorbs light at the wavelength of emission of the fluorescer, the fluorescer will transfer to the quencher chromophore the energy which would otherwise have been emitted as light. The Ullman methods all involve measurement of the decrease of fluorescence (light emission) of the fluorescer chromophore resulting from energy transfer to the quencher chromophore. Ligand and antiligand can be separately labeled with fluorescer and quencher, or one group of antibodies can be labeled with fluorescer and another group of antibodies labeled with quencher for detection of ligand capable of immunologically binding to more than one antibody.
U.S. Pat. No. 4,536,479 to Vander-Mallie discloses an immunoassay method for the detection of an analyte in a test sample wherein a reaction mixture is formed between test sample and a pair of reagents. The first reagent is an idiotypic anti-analyte antibody labeled with a first fluorophore. The second reagent is an anti-idiotypic antibody labeled with a second fluorophore, which anti-idiotypic antibody is capable of competing with analyte in the sample for the idiotypic anti-analyte antibody. One of the fluorophores is capable of absorbing incident light at a first wavelength to produce light emission at a second wavelength which second wavelength can be absorbed by the other fluorophore to produce emission at a third wavelength. For the detection of analyte in the reaction mixture, the reaction mixture is irradiated with incident light of the first wavelength, and the intensity of light of the second or third wavelength is measured, which intensity is related to the amount of analyte initially present in the test sample.
All of the known fluorescence immunoassay methods have disadvantages and limitations, leaving a continuing need for new, rapid and sensitive methods for detecting analytes in fluid samples.